PROJECT SUMMARY/ABSTRACT Somatic cell genome editing (SCGE) has remarkable promise to transform our therapeutic toolbox for the treatment of human genetic disorders. However, despite having the tools available for identification and modification of human disease-causing mutations, outstanding concerns over efficacy and safety have curtailed the clinical application of SCGE broadly. Critical gaps in our current knowledge of the safety of SCGE approaches include: 1) what are the on- and off-target genome editing rates, 2) how do SCGE reagents affect human cellular and tissue function, and 3) how will the innate and adaptive immune system respond to SCGE reagents. While clinical trials are effective tools to determine efficacy and safety, they are most efficiently applied after exhaustive pre-clinical studies have optimized efficacy and safety in other systems. The goal of this proposal is to adapt biomimetic human cardiac microtissues (CMTs)--engineered from cardiomyocytes derived from human induced pluripotent stem cells (iPSCs), fibroblasts, and macrophages--to study the impact of SCGE reagents and delivery systems on a functional human tissue. Because CMTs recapitulate in vivo cardiac three-dimensional architecture, biomechanical properties, and complex multicellular interactions that are critical to cardiac tissue homeostasis and function, they are ideal for assaying cardiac functions in vitro. The CMTs have been optimized for functional assays that quantify a range of dynamic phenotypes that include orders-of-magnitude changes in tissue contractility, calcium handling, and electrophysiology that predict in vivo cardiac function. Importantly, CRISPR/Cas9 genome editors have been effectively applied to CMTs to generate monogenic disease models of common cardiovascular disorders such as dilated and hypertrophic cardiomyopathies that result in heart failure. Guided by their comprehensive preliminary data including application of next-generation DNA- and RNA- sequencing assays to CMTs, the researchers propose to pursue two Specific Aims to determine SCGE efficacy and safety: 1) interrogate CMTs by comprehensive assessment of contractility, calcium handling and electrical function in combination with single-cell transcriptomics paired with off-target genome sequencing to identify adverse consequences of SCGE reagents that target the titin-encoding gene TTN, and 2) engineer autologous CMTs assembled with two distinct classes of macrophages to study cardiac function and SCGE reagents. Execution of these Aims will provide multi-scale insights into the safety and efficacy of SCGE reagents by producing an informative testing platform and system of associated methods to identify adverse outcomes. Establishing these resources will be a pivotal step toward realizing the promise of genome editing and human precision medicine of cardiovascular and other disorders.